Purification and enzyme kinetics of newly discovered metallo-beta-lactamase NWM-1

Question

The worldwide increase of multidrug-resistant gram-negative bacteria has become an important clinical challenge. Resistance against carbapenems is of particular concern. It can be caused by a variety of mechanisms, however the worldwide spread of carbapenemases is especially important. A worrying trend is the dissemination of Ambler class B metallo-beta-lactamases (MBL). In 2020, a carbapenem-resistant clinical P. aeruginosa isolate was referred to the National Reference Laboratory for Multidrug-resistant Gram-negative Bacteria. This isolate harboured the novel bla_NWM-1 MBL gene (North Rhine-Westphalia metallo-beta-lactamase).

Methods

The carbapenem-resistant P. aeruginosa isolate NRZ-63282 was subjected to Illumina whole-genome sequencing and the genome was screened for beta-lactamase-encoding genes. The bla_NWM-1 MBL gene was cloned into the pASG-IBA vector and transformed into E. coli JM83. Purification of NWM-1 was performed by heterologous expression in E. coli JM83 and cell disruption via sonification. The lysate was then purified via two-step fast protein liquid chromatography (FPLC), including Strep-tag affinity chromatography and gel filtration. Kinetics were measured with the Eppendorf BioSpectrometer kinetic as previously described in 50 mM HEPES buffer with 50 µM zinc sulfate (pH 7.5) with 10 mm UV cuvettes (Sarstedt, Nümbrecht, Germany) or 2 mm cuvettes (Eppendorf, Hamburg, Germany) with three independent protein preparations.

Results

The NWM-1 protein was purified successfully via affinity chromatography and gel filtration. Initial measurements of the kinetics have been carried out, but the analyses have not yet been finalized. But first results are showing hydrolysis of penicillin G, ampicillin, piperacillin, ertapenem, imipenem and meropenem, but not for aztreonam. Michaelis-Menten kinetics will be presented at DGHM & VAAM 2024.

Conclusions

The newly discovered metallo-beta-lactamase NWM-1 shows typical hydrolyzation patterns for subclass B3 MBLs. Further studies are necessary to analyze the IC₅₀ values for a variety of inhibitors, which will also be presented at DGHM & VAAM 2024.